FIG. 1.

Establishment of stable macrophage cell lines expressing HCV proteins. (A) Cell lysates were prepared from macrophage cell lines expressing each of the HCV proteins (4 × 10⁶ cells) and immunoblotted with antibodies against HCV proteins or β-actin. (B) Total RNA was extracted from macrophage cell lines expressing NS5A (gray bars) or control (white bars), and the expression of mRNA of TLRs was determined by real-time PCR. (C) The subcellular localization of NS5A was examined by confocal microscopy. Cells were fixed with 4% paraformaldehyde-PBS, permeabilized with 0.5% Triton X-100, and stained with specific antibodies. Cells expressing NS5A or control cells were extracted into cytosol (C), membrane-organelle (M), and nuclear (N) fractions. Each fraction was concentrated and subjected to immunoblotting with specific antibodies. PA28α, calregulin, and histone H1 were used as markers for cytosol, membrane-organelle, and nuclear fractions, respectively.